Two critical factors that affect the prognosis of bladder cancer patients are high-grade invasive tumors and the high rate of tumor recurrence. Hyaluronidase is an enzyme that degrades hyaluronic acid (HA), a glycosaminoglycan, into small angiogenic fragments. Two hyaluronidases have been identified as potential "molecular markers" of high-grade bladder tumors (BT). The long-term objectives of this proposal are to elucidate functions of these hyaluronidases in BT progression and to develop a simple, non-invasive and accurate test for early detection and post-therapy surveillance of bladder cancer. To test the hypothesis that BT-derived hyaluronidases are structurally distinct from other known hyaluronidases and are expressed in a tumor, tissue, or cell specific manner, BT-derived hyaluronidases will be isolated, purified, cDNA cloned and sequenced (Aim 1). These enzymes will be characterized for substrate specificity and sensitivity to inhibitors and their expression will be examined at protein and mRNA levels in normal tissues, tumor tissues, and tumor cells (Aim 2). To test whether elevated BT-derived hyaluronidase levels confer metastatic phenotype to otherwise indolent BT cells, low-grade BT cells will be transfected with cDNAs of these enzymes and following orthotopic implantation in nude mice metastasis will be examined. In addition, the role of angiogenic HA fragments on functions of BT cells and BT-derived endothelial cells which regulate BT progression will be examined (Aim 3). Urinary HA levels are elevated in BT patients and urinary hyaluronidase levels are elevated in patients with high-grade BT. To test whether a HA-hyaluronidase test can detect bladder tumor recurrence and indicate its malignant potential, urinary HA-hyaluronidase levels in 150 BT patients will be measured at scheduled surveillance visits and results will be compared with cystoscopic or urine cytology findings (Aim 4). The proposed study could result in identifying and functionally characterizing a new marker of high-grade BT which could improve diagnosis and treatment of bladder cancer.